Toxicity and Genotoxicity of Beauty Products on Human Skin Cells in Vitro

linica f C l To o x l ic a o n Alnuqaydan and Sanderson, J Clin Toxicol 2016, r l o u

o y

J 6:4 Journal of Clinical Toxicology DOI: 10.4172/2161-0495.1000315 ISSN: 2161-0495

Research Article Open Access Toxicity and Genotoxicity of Beauty Products on Human Skin Cells In Vitro Abdullah M Alnuqaydan* and Barbara J Sanderson Department of Medical Biotechnology, School of Medicine, Flinders University, Australia *Corresponding author: Abdullah M Alnuqaydan , Department of Medical Biotechnology, School of Medicine, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia, Tel: +61-08-7221-8556; Fax: +61-08-7221-8555; E-mail: [email protected] Received date: June 19, 2016; Accepted date: August 30, 2016; Published date: August 31, 2016 Copyright: © 2016 Alnuqaydan AM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Background: We use beauty products in high quantities every day and in the process, we are exposed to a wide variety of chemicals used in these products. These chemicals are a particularly insidious form of body pollution because they enter the human body through multiple routes. The problem with commercial products and particularly beauty products is that millions of people apply beauty products to their skin daily for long time.

Objective: To determine the toxicity and genotoxicity effects of four facial beauty products on tow human skin cells. Also, to find out which product ingredients can induce the most toxicity and genotoxicity on human skin cells.

Methodology: The in vitro toxicity and genotoxicity of facial beauty products were determined using a human keratinocyte cell line (HaCaT) and a human fibroblast cell line (CCD-1064SK). The products were an Anti-aging face moisturiser with mixture of natural ingredients (Facial Moisturizer - Camellia & Geranium Blossom) and Nivea Visage Q10plus Anti-Wrinkle which includes synthetic chemicals with TiO2 and Nivea Visage Q10plus Anti-Wrinkle which includes synthetic chemicals without TiO2. Glycerol was the negative control. Toxicity was measured by Crystal violet assay and Methyl tetrazolium cytotoxicity (MTT) assay. Apoptosis/necrosis proportion, nuclear division index (NDI) and genotoxicity were detected by cytokinesis block micronucleus (CBMN) assay.

Results: Glycerol did not induce any toxicity or genotoxicity. Nivea Visage Q10plus Anti-Wrinkle which includes synthetic chemicals with and without TiO2 showed significant toxicity in both assays. No toxicity observed with Facial Moisturizer - Camellia & Geranium Blossom but there was a significant necrosis. Populations of cells treated with diluted Nivea Visage Q10plus Anti-Wrinkle which includes synthetic chemicals with and without TiO2 showed increased proportions of apoptosis/necrosis. The nuclear division index (NDI) was decreased by Nivea Visage Q10plus Anti-Wrinkle which includes synthetic chemicals with and without TiO2 and Facial Moisturizer - Camellia & Geranium Blossom. Nivea Visage Q10plus Anti-Wrinkle which includes synthetic chemicals with and without TiO2 showed increased frequency of micronuclei (MNi). Nivea Visage Q10plus Anti-Wrinkle face moisturizer with TiO2 proved to induce significantly more micronuclei (MNi) than the product without TiO2.

Conclusion: The study results indicate that facial beauty products can cause cytotoxicity and genotoxicity in vitro using dilutions of the commercial formulations.

Keywords: Toxicity; Genotoxicity; Safety assessment; Beauty companies that make them are self-regulating, and government products; Cosmetic ingredients; Cell culture; Chromosomal damage agencies do not press the manufacturers to prove their products are safe [1,2]. In the US, cosmetic and personal care products are not Introduction regulated by the Food and Drug Administration (U.S. FDA) [3,4]. However, drugs do require extensive testing and approval by the FDA We use large quantities of beauty products every day and, in the [3]. Also, one study has noted the results of studies screening blood process, are exposed to a wide variety of chemicals used in these sample from over the entire world, indicate that most people are products. These chemicals are a particularly insidious form of body carrying a huge amount of chemicals in their bodies [1,5]. These pollution because they enter the human body through multiple routes. studies used biochemical methods for screening. Another study have It is easy to swallow them, inhale them and absorb them through the shown that exposure to chemicals demonstrates that most American mucous membrane of the eyes, mouth or nose. Our skin absorbs children and adults carry inside them nearly 100 substances or approximately 60% of the chemical ingredients and sends them into chemicals including pesticides and toxic compounds [5]. Many of the bloodstream, from whence they can reach every organ in the body these cause cancer, damage the immune system and affect human seconds after absorption [1]. Women using a lot of cosmetics are behaviour and the central nervous system. The sources of these thought their skin absorb up to 2 kg of chemical cosmetic ingredients chemicals include household exposure to pesticides and detergents, each year [1]. Government reports in the US and EU indicate that cosmetics, toiletries, paints and fabric treatments [1,2,6]. They can about 90% of the ingredients used in cosmetics are not safe for people affect the body over the long term and accumulate in different organs in the long-term [1]. Most beauty products contain a mixture of and the bloodstream and then pass through the urine, semen and in chemicals that only make the problem worse [2]. Unfortunately, the the form of breast milk. After a while and the body becomes

J Clin Toxicol, an open access journal Volume 6 • Issue 4 • 1000315 ISSN:2161-0495 Citation: Alnuqaydan AM, Sanderson BJ (2016) Toxicity and Genotoxicity of Beauty Products on Human Skin Cells In Vitro. J Clin Toxicol 6: 315. doi:10.4172/2161-0495.1000315

Page 2 of 9 overloaded and at risk of total breakdown [1]. Some cosmetics contain moisturizers with sun blocks contain Titanium dioxide (TiO2) which is mercury which is used to lighten the skin and people who use products a potential hazard and carcinogen [11,12]. Finally, most shampoos and containing mercury are at a high risk of mercury poisoning [7]. In the other toiletries or liquid formulas contain nitrosamines that can cause United States mercury compounds are used as preservatives in small cancer [13]. Some products are labelled as hypoallergenic but probably concentrations for eye area products and FDA regulations in the US still contain potentially carcinogenic substances [14]. In this study, four have restricted cosmetics products that contain mercury [7]. Some different facial beauty products were examined to assess the effects on moisturizers contain mineral oil which can slow down cell renewal and two human skin cells (Human keratinocytes HaCaT skin cells and promote early skin ageing [1]. A study tested 88 brands of eye shadow human fibroblast CCD-1064SK cells). Products were Nivea Visage Q10 and found that approximately 75% of these products contained at least Plus Anti-Wrinkle face moisturizer which includes synthetic chemicals one of the 5 elements: lead, nickel, chromium, arsenic or cobalt [8]. + TiO2, Nivea Visage Q10 Plus Anti-Wrinkle face moisturizer which Lead can damage any part of the human body and in particular the includes synthetic chemicals (Improved formula, without TiO2), Facial nervous system [9]. Even the elements found in small doses in these Moisturizer - Camellia & Geranium Blossom which includes a mixture products may cause hormone disruption [10]. Some sun blocks and of natural ingredients and Glycerol B.P.

Ingredients Toxic effects

Octocrylene Skin allergen. Restricted for use in cosmetics in Japan. Produces excess ROS that can interfere with cellular signalling, cause mutations, lead to cell death and may be implicated in cardiovascular disease. Measured to accumulate in people.

Ethylhexyl Salicylate Low allergies and immunotoxicity, ecotoxicology

Methylpropanediol Not expected to be potentially toxic or harmful

Glyceryl Stearate Suspected to be an environmental toxin

Butyl Methoxydibenzoylmethane Toxin in mice

C12-15 Alkyl Benzoate Suspected to be an environmental toxin

Tocopheryl Acetate Human skin toxin or allergen—strong evidence. Has caused tumours in animals.

Chondrus Crispus Organ system toxin (non-reproductive)

Dimethicone Organ system toxin (non-reproductive)

Trisodium EDTA Penetration enhancer

Caprylic/Capric Triglyceride Ecotoxin

Limonene and Parfum Irritant. Possible human immune system toxin or allergen. Restricted in cosmetics

Ingredients Toxic effects

Methylparaben Human endocrine disruptor-strong evidence

Phenoxyethanol Irritant (skin, eyes or lungs), occupational hazard, organ system toxin (non-reproductive)

Cera Microcristallina Organ system toxin (non-reproductive)

Paraffinum Liquidum Human immune and respiratory toxin or allergen—strong evidence

Benzyl Alcohol Occupational hazard, organ system toxin (non-reproductive)

TiO2 Carcinogen

Thylhexylglycerin Irritant (skin, eyes or lungs); organ system toxin (non-reproductive)

Carbomer No carcinogenicity data available, but it is found to be irritating to the respiratory tract.

Sodium Phenylbenzimidazole Sulfonat May cause skin irritation, if swallowed will cause vomiting.

Trimethoxycaprylylsilane Not expected to be potentially toxic or harmful

Table 1: The ingredients and toxic effects of Nivea Visage Q10Plus Anti-Wrinkle face moisturizer + TiO2. The toxic effects of the ingredients were classified by [39-41].

Page 3 of 9 Materials and Methods soften and moisturize the skin. Glycerol may reduce food intake in diabetic rats [17]. Therefore, there is a label on the package warning Materials the diabetic patient. Glycerol is a common basic ingredient in many moisturizers. Therefore, it used as a negative control for the beauty RPMI 1640 media and foetal bovine serum (FBS) were purchased products experiments. from Gibco® Cell Culture Media - Life Technologies (Australia). Cytochalasin B (Cyt-B) solution, Sodium dodecyl sulphate (SDS, Cell lines and cell culture approximately 99%), Phosphate buffered saline (PBS) and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were A human non-cancer keratinocytes cell line HaCaT were a gift from purchased from Sigma-Aldrich (USA). Spectrophotometer plate reader the Department of Haematology and Genetic Pathology-Flinders (BIO-TEK Instruments Inc., USA). Diff-Quik stains were purchased Medical Centre, School of Medicine at Flinders University, Adelaide. from Lab Aids (Australia). Cytospin centrifuge (Shandon, England). Skin fibroblast cell line CCD-1064Sk (A human normal skin cells) was ® ™ TrypLE™ was purchased from Life Technologies (Australia). All other obtained from ATCC, US (ATCC CRL-2076 ). Keratinocytes cell line reagents were obtained from sigma, unless otherwise stated. HaCaT was maintained in RPMI 1640 medium, with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Scientific, Products to be examined Australia). A human normal skin fibroblast cell line CCD-1064Sk was maintained in Iscove's Modified Dulbecco's medium (IMDM medium) Nivea visage Q10Plus Anti-wrinkle face moisturiser with titanium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. dioxide (TiO2) Cells were seeded in tissue culture flasks and incubated at 37°C in a 5% CO fully humidified incubator. HaCaT cells were subcultured when Nivea visage Q10plus Anti-Wrinkle day moisturizer cream plus 2 they reached 60–80% confluence. extra UVA protection (SPF 15) is produced by Nivea which is a worldwide company. Nivea Visage Q10plus Anti-Wrinkle purchased from a local pharmacy in Adelaide, South Australia. It suits all skin Cell treatment types. The product aims to increase the natural Q10 level and prevent The 96_well flat bottom was seeded with 104 cells/well and wrinkles. Also, it is protecting from UVA+UVB. The ingredients of incubated for 19 h to allow the adherence of cells at 37°C in 5% CO2. Nivea Visage Q10plus are mixture of chemicals (Table 1). The media were aspirated and replaced with 100 μL of the treatment solution per well and were treated for 1 h prior to bioassays or genetic This formula of the product contains Titanium Dioxide (TiO2) which is a nanoparticle that is used widely in pigments, cosmetics, and assays. The negative or untreated control (0 dose) was the media. skin care products because it the benefit of protecting the skin from UV light, particularly in Nano sized particles less than 100 nm [15]. Crystal violet assay Titanium Dioxide (TiO ) has been classified as carcinogen [16]. Some 2 Crystal violet stains the DNA of the live cells that adheres the plate studies have shown that Titanium Dioxide TiO can damage DNA 2 after the dead cells are washed away [18]. The relative number of viable directly or indirectly via inflammatory response or oxidative stress cells was determined using crystal violet assay (CV) as described in [15]. [19]. Briefly, 50 µL of crystal violet stain (0.5% of crystal violet in 50% Nivea Visage Q10Plus Anti-Wrinkle face moisturizer (Improved methanol) was added to each well and incubated for 10 minutes at formula, without titanium dioxide (TiO2) ambient temperature. After 10 minutes, the plate was gently washed with distilled water then air dried. 50 µL of 33% acetic acid was added This product is an improved formula of Nivea Visage Q10plus Anti- to de-stain the cells. The absorbance (ODs) was measured on a Wrinkle day moisturizer. It is released into the market after removed spectrophotometric plate reader using a test wavelength of 570 nm the original product which contains Titanium Dioxide TiO . It has 2 with a reference wavelength of 630. almost the same ingredients as the original one except for the absence of TiO2. The Package is labelled with ‘skin compatibility dermatologically approved'. This product aims to UVA protection. The Methyl tetrazolium cytotoxicity assay (MTT Assay) product is suitable for sensitive skin. The tetrazolium salt 3-4,5 dimethlthiazol-2,5 diphenyl tetrazolium bromide (MTT) assay is based on a colorimetric assay for mammalian Grown facial moisturizer - camellia & geranium blossom cell growth and survival, and it depends on the ability of viable cells to metabolize the yellow and water-soluble tetrazolium salt [20]. Cells Facial Moisturizer - Camellia & Geranium Blossom is a natural were seeded at 104 in a volume of 100 µl into each well of a 96-well flat moisturizer made from bioactive ingredients. It is made by extractions bottom plate. MTT solution with a final concentration 0.5 mg/ml was from Camellia and Rose Hip Seed Oil which consists of vital added and then incubated for 4 h at 37°C. After incubation, 80 µl 20% phytosterols that rehydrate and nourish the skin. Cane sugar is also SDS in 0.02 M HCl was added. The plates were incubated overnight in present, and it releases bio saccharides that soothe the skin and the dark at room temperature. Thee absorbance (ODs) was measured combats the effects of UV and pollution while Mayblossom releases on a spectrophotometric plate reader using a reference wavelength of flavonoids which normalize sebum production and reduces pore size. 630 nm and a test wavelength of 570 nm. The product was purchased from a local chemist. Cytokinesis block micronucleus assay (CBMN Assay) Glycerol British Pharmacopoeia B.P. The mechanism of cell killing and genotoxicity of beauty products Pharmaceuticals Pty Ltd produces glycerol B.P. It was purchased was carried out using Cytokinesis-Block Micronucleus Assay (CBMN) from a local pharmacy. It is 90-100% Glycerol (Glycerine). It can be assay as described [21,22]. Briefly, after treatment Cyt-B (4.5 µg/ml) prescribed to be taken internally as a mild laxative and externally to

Page 4 of 9 was added to the media and the cultures were incubated at 37ºC for 23 0.03% w/v doses of Nivea visage without TiO2 after treated HaCaT cells h. Cells were trypsinized (TrypLE™ Express Enzyme (1X), phenol red) for 1 h. Otherwise, no significantly apoptosis or necrosis induction was and collected onto slides by a cytospin centrifuging for 5 minutes at 47 observed in the treatments of Facial natural treatment (Facial ×g (@6000 rpm). Slides were air-dried, fixed by DiffQuick Fixative for Moisturizer - Camellia & Geranium Blossom) and Glycerol B.P on 10 min, and then double stained with stain 1 (red DiffQuick Stain) and HaCaT cells as shown in Figure 3A. then Stain 2 (blue DiffQuick Stain). Slides were scored as described in [23]. The chromosomal damage induced by treatment and total number of micronuclei (MNi) in binucleated (BN) cells totalled 1000. Slides were scored at a magnification of 250X or 40 X. Criteria for scoring micronuclei MNi, nucleoplasmic bridge (NPB) or nuclear buds (NBUDs) were as described [21]. Cytotoxicity determination induced by treatment, and the percentage of apoptosis/necrosis were evaluated in 500 cells and calculated according to published formulae [23,24].

Statically analysis Data were presented as the mean ± S.E.M. of the standard error. The experiments were replicated at least three independent times. Statistical analysis of the data was carried out using ANOVA, followed by Tukey’s HSD post hoc test. These tests were performed using SPSS software (Version 22). Differences were considered significant when the p-value was less than 0.05. Responses to treatment were compared to the untreated control (0 doses) which is represented as 100% survival.

Results

Cytotoxicity effects of beauty products on human skin cells The toxicity of four beauty products on Keratinocytes human skin cells (HaCaT) and human normal skin fibroblast (CCD-1064Sk) in vitro was determined by incubating cells with treatments for 1h. Two cytotoxicity assays were carried out to indicate the toxicity of beauty products. The MTT cell survival assay was used to determine the relative survival cells when yellow MTT is reduced to purple formazan in the mitochondria of living cells. The Crystal Violet (CV) assay was used to determine the relative cell number when Crystal violet stains the DNA of the live cells that adhere to the plate after the dead cells are washed away. There was significant toxicity with doses of 3% w/v and 0.3% w/v for Nivea Visage Q10plus Anti-Wrinkle face moisturizer (with TiO2) after treated HaCaT cells for 1h (Figure 1). Also, significant toxicity was induced by the highest dose (3% w/v) of Nivea Figure 1: Relative cell viability and cell number (%) after treatment Visage Q10plus Anti-Wrinkle face moisturizer (Improved formula, of HaCaT human skin cells with (A) Glycerol B.P, (B) Facial without TiO2) on human fibroblast CCD-1064SK cells determined by Moisturizer - Camellia & Geranium Blossom and (C) Nivea Visage MTT and Crystal Violet (Figure 2). However, no significant toxicity Q10plus Anti-Wrinkle face moisturizer (with TiO2) for 1 hour. emerged on HaCaT human skin cells or human fibroblast Relative survival was measured by the MTT assay. Relative cell CCD-1064SK cells when using treatments of Glycerol B.P or Facial number was measured by the crystal violet assay. Data are shown as Moisturizer - Camellia & Geranium Blossom. a percentage compared to untreated control and are mean of three replicates ± standard error of the mean (S.E.M). Treatments The Nuclear division index (NDI) is a method employed to measure significantly different from untreated control at P<0.05 are the proliferative status of viable cells that can be used to assess general presented as ‘*’. toxicity [21,25]. Table 2 shows the value of NDI for all beauty products examined and which had a significantly lower NDI value in the highest dose (3% w/v; 1.4 (P<0.05) of Nivea Visage with or without TiO2 and Facial Moisturizer - Camellia & Geranium Blossom treatment at dose 5% w/v; 1.4 P<0.05 on HaCaT cells.

Mechanism of cell killing The CBMN results (Figure 3 B) detected a significant increase in late apoptosis and early necrosis induced by the highest dose of Nivea visage with TiO2 3% w/v and significantly induced in 0.3% w/v and

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Figure 3: Apoptosis and necrosis induction detected by the CBMN assay for HaCaT cells followed by 1 h treatment using (A) Facial natural treatment (Facial Moisturizer - Camellia & Geranium Blossom) and Glycerol B.P and (B) using Nivea Visage Q10plus Anti-Wrinkle face moisturizer with TiO2 and Nivea Visage Q10plus Anti-Wrinkle face moisturizer (Improved formula, free of TiO2). Treatment Data are shown as the mean of three observations ± SEM. Treatments significantly different from untreated control at P<0.05 are presented as ‘*’.

Figure 2: Relative cell viability and cell number (%) after treatment of CCD-1064Sk normal fibroblast human skin cells with (A) Glycerol B.P, (B) Facial Moisturizer - Camellia & Geranium Blossom and (C) Nivea Visage Q10plus Anti-Wrinkle face moisturizer (Improved formula, without TiO2) for 1 hour. Relative survival was measured by the MTT assay. Relative cell number was measured by the crystal violet assay. Data are shown as a percentage compared to untreated control and are mean of three replicates ± standard error of the mean (S.E.M). Treatments significantly different from untreated control at P<0.05 are presented as ‘*’.

Nivea visage without TiO induced significant late apoptosis and 2 Figure 4: Apoptosis and necrosis induction detected by the CBMN early necrosis on fibroblast cells (CCD-1064SK) at dose 0.03% w/v and assay for CCD_1064SK cells followed by 1 h treatment using Nivea significant early necrosis induced by Facial Moisturizer - Camellia & Visage Q10plus Anti-Wrinkle face moisturizer (improved formula, Geranium Blossom at dose 5% w/v on CCD 1064SKas shown in figure without TiO 0.3% w/v and 0.03% w/v and Facial Moisturizer - 4. However, no significant induction of apoptosis or necrosis was 2 Camellia & Geranium Blossom 5% w/v and 0.5% w/v and Glycerol observed on the the tretment of Glycerol B.P on CCD-1064SK cells. B.P 10% v/v and 1% v/v. Data are shown as the mean of three Finally, this result was consistent with the previous result of Nivea observations ± SEM. Treatments significantly different from Visage at dose 3% w/v induced necrosis detected by apoptosis assay untreated control at P<0.05 are presented as ‘*’. followed flow cytometry (data not shown).

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Genotoxicity of beauty products on human skin cells TiO2 was significantly difference from the product of Nivea Visage without TiO and from untreated control. This result means that the The genotoxicity of beauty products on HaCaT human skin cells 2 product of Nivea Visage which contains TiO2 cause significant and CCD_ 1064SK was carried out using Cytokinesis-Block gentoxicicity on HaCaT cells compare to the product of Nivea Visage Micronucleus Assay (CBMN) assay. The following measures of removed TiO2 showed less genotoxic on HaCaT cells. Nucleoplasmic genotoxicity chromosome breakage and chromosome loss bridge (NPB) and Nucleoplasmic buds (NBUDs) were also observed in (micronucleus MNi), chromosome rearrangement (nucleoplasmic the products of Nivea Visage Q10plus Anti-Wrinkle face moisturizer + bridges) and gene amplification (nuclear buds) [26]. The frequency of TiO2 and Facial Moisturizer - Camellia & Geranium Blossom (natural the induced micronuclei (MNi) indicates the extent of chromosomal product) but they did not reach a significant level. A significant changes induced by beauty products. The result of CBMN assay increase in micronucleus (MNi) was observed at 0.3% w/v and 0.03% showed the genotoxicity effects of beauty products on HaCaT cells as w/v doses of Nivea Visage Q10plus Anti-Wrinkle face moisturizer shown in Figures 5. No significant increase in MNi observed in the (Improved formula, without TiO2) after treated CCD_1065SK cells results of treated HaCaT cells with Glycerol B.P and Facial Moisturizer (Figure 6). Other treatments demonstrated an increase in the number - Camellia & Geranium Blossom (natural product) for 1 h (Figure 5 of MNi but they did not reach a significant level (Figure 6). A). Nucleoplasmic bridge (NPB) and Nucleoplasmic buds (NBUDs) were not observed in all products treatments on CCD_1065SK cells.

Figure 6: The frequency of micronuclei (MNi) per 1000 binucleated cells was determined using the Cytokinesis Block Micronucleus (CBMN) assay following exposure of CCD_1065SK cells to Nivea Visage Q10plus Anti-Wrinkle face moisturizer (Improved formula, without TiO2) treatment, Glycerol B P, Facial Moisturizer - Camellia & Geranium Blossom (natural product). The data are the mean ± S.E.M. from three separate experiments. Treatments significantly different from untreated control at P<0.05 are presented as ‘*’.

Figure 5: Frequency of micronucleated binucleate (MNBNCs) as Discussion measured by the CBMN assay following exposure of HaCaT cells to (A) Glycerol B.P and Facial Moisturizer - Camellia & Geranium In this study, two human normal skin cell lines were used to Blossom (natural product) and (B) Nivea Visage Q10plus Anti- examine the toxicity and genotoxic effects of beauty products in vitro. Wrinkle face moisturizer with TiO2 and Nivea Visage Q10plus Anti- Human keratinocyte cell line (HaCaT) which is derived from full Wrinkle face moisturizer (Improved formula, without TiO2) epidermal differentiation capacity, functions as the outermost layer of treatment. The data are the mean ± S.E.M. from three separate the skin [27-29]. Human dermal fibroblast cells CCD-1046 within the experiments. Treatments significantly different from the untreated dermis layer of skin are responsible for generating connective tissue control at P<0.05 are presented as ‘*’ or ‘¥’ Treatments differed [27,30]. Glycerol B.P served as a negative control in this study because significantly from Nivea Visage Q10plus Anti-Wrinkle face it is a common basic ingredient in many moisturizers. There was no moisturizer with TiO2. cytotoxic effect of exposure 1h to Glycerol B.P on HaCaT or CCD 1064SK cells. The Nuclear division index (NDI) obtained from the CBMN assay provides a measure of cell division [31]. There was no However, there was significant increase in the number of MNi at the significant change in the NDI value which reflects the fact that dose 0.3% w/v of Nivea Visage Q10plus Anti-Wrinkle face moisturizer Glycerol B.P did not affect the cell cycling in both cell lines. + TiO2. Also, Figure 5B showed the result of HaCaT cells after treated Furthermore, the evaluation of apoptosis and necrosis of Glycerol B.P with two different formulas of Nivea visage product (Nivea Visage on human skin cells detected by CBMN assay showed no significant Q10plus Anti-Wrinkle face moisturizer with TiO2 and Nivea Visage difference from the untreated control. Also, the frequency of micro Q10plus Anti-Wrinkle face moisturizer without TiO2. There was nucleated binucleate (MNi), nucleoplasmic bridges (NPBs) and significant difference between the two formulas of Nivea Visage nuclear buds (NBUDs) were not observed in 1000 binucleated (BN) products on HaCaT cells. The product of Nivea Visage which contains cells which indicated that no genetic damage occurred after treatment

Page 7 of 9 with Glycerol. Therefore, Glycerol is a safe ingredient used in several to untreated control and are mean of 3 replicates, standard error of the cosmetics products. The Facial Moisturizer - Camellia & Geranium mean ± (S.E.M). A significant difference from untreated control at Blossom treatment used in this study is a product of mixture of natural *P<0.05. ingredients listed on the product. It showed no significant toxicity compared to the untreated control on CCD_1064SK cells. Consistent Titanium dioxide (TiO2) which is a nanoparticle that is classified as with this result is the fact that there was no cytotoxicity observed on a carcinogen [15]. Titanium dioxide (TiO2) plays a role in the treated HaCaT cells with Facial Moisturizer - Camellia & Geranium induction of apoptosis as well as oxidative stress. Moreover, studies Blossom treatment for 1h (Figure 1). The Nuclear division index have shown that Titanium dioxide (TiO2) causes genetic damage linked (NDI), based on the result of the CBMN assay, revealed a significant to DNA-adduct formation in human lung cells [34-36]. The metabolic decrease in dose 5% w/v lower NDI value 1.4 (P<0.05) in HaCaT cells. effects of Titanium dioxide (TiO2) on keratinocytes HaCaT cells have This means a change occurred in the rate of cell cycling - they took also being investigated. One study indicated that Titanium dioxide longer to divide, or the viable cells failed to divide during the affect the mitochondria [11]. Another study demonstrated a significant cytokinesis-block [21]. No apoptosis or necrosis induction was uptake of TiO2 in keratinocytes in human skin cells (HaCaT); this was observed after HaCaT cells were treated with Facial Moisturizer - performed using transmission electron microscopy (TEM) and flow Camellia & Geranium Blossom treatment for 1h at both doses. cytometry [37]. However, a small but statistically significant necrosis was observed in In our studies Nivea Visage Q10plus Anti-Wrinkle face moisturizer CCD-1064Sk cells at the higher (5% w/v) dose of the Facial with TiO was compared to an identical product without the TiO Moisturizer - Camellia & Geranium Blossom treatment after treatment 2 2 allowing for an evaluation of the effect of the TiO . The result of MTT for 1h. 2 and Crystal Violet showed significant toxicity with the two doses of Furthermore, the CBMN result indicated that no chromosomal Nivea visage +TiO2 was noted- up to 76% and 92% cells were killed damage causes by Grown Facial Moisturizer - Camellia & Geranium after a 1 h exposure to a 0.3% and 3% (w/v) dose, respectively (Figure Blossom after human skin cells were treated for 1h. 1). Grown Facial Moisturizer - Camellia & Geranium Blossom The mechanism of cell death was elucidated using the CBMN assay. treatment did not demonstrate significant toxicity or genotoxicity in There was a significant induction of late apoptosis and early necrosis at human skin cell lines, but this does not mean it is a safe product to use. (0.3% w/v) on HaCaT human skin cells (Figure 3B). Also, a significant The change in NDI value indicates the decrease in the cell cycle was 1.4 low NDI value (1.4 (P<0.05)) was observed at the 3% w/v dose (Table (P<0.05) on the HaCaT cell line. Also, significant necrosis observed 2). after treated fibroblast cell lines with Facial Moisturizer - Camellia & Also, nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) Geranium Blossom for 1h at dose (5% w/v). were observed. These outcomes illustrate some of the mechanisms of Green or botanical products are not well regulated by government chromosome damage when using Nivea Visage Q10plus Anti-Wrinkle agencies. There is advice to avoid products that use essential oils such day moisturizer. The frequency of chromosome rearrangement is as lavender oil or tea tree oil that are classified as hormone disruptors indicated by NPB and NBUDs. NPB may arise from dicentric [32,33]. chromosomes and NBUDs from gene amplification [21]. A dicentric chromosome and an acentric chromosome fragment are formed as a NDI value result, and they manifest themselves in the formation of an NPB and

Treatments HaCaT cell line CCD_1064SK cell line MN [21]. The formation of NPBs could lead to misrepair of DNA strand breaks which could also lead to a dicentric chromosome and Media control 1.8 1.6 concatenated ring chromosome. One dicentric chromosome mechanism could result in telomere end fusion which is caused via NVAW + TiO2 dose 0.3% w/v *1.4 - shortening or loss of the telomere capping protein [21]. This study is

NVAW + TiO2 dose 0.03% w/v 1.7 - consistent with that conducted by [38] which demonstrated that sunscreens containing Titanium dioxide can catalyse oxidative damage NVAW without TiO2 dose 0.3% *1.4 1.5 to DNA in vitro and in human cell culture. w/v On the other hand, Nivea Visage Q10 plus Anti-Wrinkle face NVAW without TiO2 dose 0.03% 1.6 1.5 moisturizer (Improved formula, without TiO ) showed significant w/v 2 toxicity on CCD-1064SK at (3% and 0.3% w/v) measured by MTT and FMCGB treatment dose 5% w/v *1.4 1.5 Crystal Violet assays. The mechanism of cell death scored by the CBMN assay (Figure 4) showed there was significant induction of FMCGB treatment dose 0.5% 1.6 1.6 apoptosis and necrosis at (0.03% w/v) on CCD-1064. w/v Genetic damage effects detected by the CBMN assay showed a Glycerol dose 10% v/v 1.7 1.6 significant increase in MNi (28.3 MNi/1000 binucleated cells, n=3) Glycerol dose 1% v/v 1.8 1.6 (P<0.05) in CCD_1064SK cell lines. However, there was no significant increase in MNi with HaCaT cells as (Figure 5B). Also, a significant low NDI value (1.4 (P<0.05)) was observed only in 3% w/v dose on Table 2: Nuclear Division Index (NDI) comparison between untreated HaCaT cells (Table 2). This means that the cells took a longer time to control (0 doses) and beauty products. Cells were plated and described divide in HaCaT cells after being treated with Nivea Visage Q10plus in the materials and methods section. Cells exposed to beauty Anti-Wrinkle face moisturizer (Improved formula, without TiO ) for treatments for 1h. NDI was determined by the cytokinesis-block 2 micronucleus (CBMN) assay. Data are shown as percentages compared

Page 8 of 9 1h. Nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) were References not observed on CCD_1064SK. 1. Thomas P (2008) Skin Deep: The Essential Guide to What's in the This product consists of a mixture of chemicals ingredients that are Toiletries and cosmetis you use. Rodale an imprint of Pan Macmillan the same as those reported in Table 1, except Titanium dioxide which 2. Zeliger H (2008) Human toxicology of chemical mixtures: toxic was removed from this improved formula that released into the market consequences beyond the impact of one-component product and by Nivea Company. Therefore, the even though none of these environmental exposures. William Andrew. chemicals individually are known to be carcinogenic it is apparent that 3. Lazarus MC, Baumann LS (2001) The use of cosmeceutical moisturizers. the mixture has shown carcinogenicity [2]. It is hypothesized that Dermatologic Therapy 14: 200-207. chemicals in mixtures could interact with each other and become 4. U.S.FDA (2006) Cosmetics. In USFaD Administration, PaPY Health eds carcinogenic. A brain cancer cluster study concluded that different US. mixtures of chemicals can induce the same cancer types despite using 5. Cone M (2005) Dozens of Chemicals Found in Most Americans' Bodies. different mechanisms. None of the chemicals are known to The Los Angeles Times A 21. individually cause brain cancer [2]. 6. Menegaux F, Baruchel A, Bertrand Y, Lescoeur B, Leverger G, et al. (2006) Household exposure to pesticides and risk of childhood acute leukaemia. Interestingly, Nivea Visage Q10plus Anti-Wrinkle face moisturizer Occup Environ Med 63: 131-134. with TiO2 proved to induce significantly more micronuclei than the 7. Balluz LS, Philen RM, Sewell CM, Voorhees RE, Falter KH, et al. (1997) Mercury toxicity associated with a beauty lotion, New Mexico. Int J product without TiO2. As mentioned earlier, the difference is Epidemiol 26: 1131-1132. compatible with findings that TiO2 enters the nucleus and cytoplasm of keratinocytes causing oxidative stress damage to DNA [37]. 8. Sainio EL, Jolanki R, Hakala E, Kanerva L (2000) Metals and arsenic in eye shadows. Contact Dermatitis 42: 5-10. Consumers who are using Nivea Visage Q10plus Anti-Wrinkle day 9. Järup L (2003) Hazards of heavy metal contamination. Br Med Bull 68: moisturizer are in fact exposed to an undiluted product (100%) with 167-182. the potential to create long-term damage. Carcinogens in cosmetics 10. Rubin BS (2011) Bisphenol A: an endocrine disruptor with widespread and personal care products are potentially greater cancer risks than exposure and multiple effects. J Steroid Biochem Mol Biol 127: 27-34. food contaminated with industrial carcinogens or pesticides because 11. Tucci P, Porta G, Agostini M, Dinsdale D, Iavicoli I, et al. (2013) chemicals ingested into the body by mouth are absorbed by the Metabolic effects of TiO2 nanoparticles, a common component of intestines and pass into the venous blood. These chemicals are then sunscreens and cosmetics, on human keratinocytes. Cell Death Dis 4: transported to the liver which exists to detoxify the substances to e549. varying degrees by enzymes before they can reach the rest of the body 12. Falck GC, Lindberg HK, Suhonen S, Vippola M, Vanhala E, et al. (2009) Genotoxic effects of nanosized and fine TiO2. Hum Exp Toxicol 28: [33]. However, carcinogens absorbed by the skin can bypass the liver 339-352. and circulate through the blood stream, thus reaching every organ in 13. King AG (2011) Research Advances: Addressing the Environmental Fates the body [1,33]. of Everyday Products from Cigarette Butts to Shampoos and Cleaning In conclusion, the current study has shown the possible harmful Agents. Journal of Chemical Education 88: 8-8. effects of several beauty products on normal human skin cells in vitro. 14. Millikan LE (2001) Cosmetology, cosmetics, cosmeceuticals: definitions In particular, the anti-aging face moisturizer which has a synthetic and regulations. Clin Dermatol 19: 371-374. chemical product (Nivea Visage Q10plus Anti-Wrinkle day moisturizer 15. Trouiller B, Reliene R, Westbrook A, Solaimani P, Schiestl RH (2009) +TiO ) induced the highest toxicity and genotoxicity of the beauty Titanium Dioxide Nanoparticles Induce DNA Damage and Genetic 2 Instability In vivo in Mice. Cancer Research 69: 8784-8789. products tested. Also, Nivea Visage Q10plus Anti-Wrinkle day 16. Zhao J, Bowman L, Zhang X, Vallyathan V, Young S-H, et al. (2009) moisturizer without TiO2 induced significant toxicity and genotoxicity Titanium dioxide (TiO2) nanoparticles induce JB6 cell apoptosis through on human fibroblast CCD_1064Sk cells. On the other hand, the face activation of the caspase-8/Bid and mitochondrial pathways. Journal of moisturizer containing natural ingredients (Facial Moisturizer - Toxicology and Environmental Health, Part A 72: 1141-1149. Camellia & Geranium Blossom) was a relatively less toxic product 17. Brief DJ, Davis JD (1982) Glycerol reduces food intake in diabetic rats. compared to other beauty products, and Glycerol B.P. (the negative Physiol Behav 29: 577-580. control) showed no toxic effect in either human cell line. Finally, 18. Berry JM, Huebner E, Butler M (1996) The crystal violet nuclei staining further investigation could be done to study specific chromosome technique leads to anomalous results in monitoring mammalian cell damage occurred by Nivea visage using fluorescent probes by cultures. Cytotechnology 21: 73-80. fluorescence in situ hybridization (FISH). Also further work could be 19. Ramezanpour M, Burke da Silva K, Sanderson B (2012) Differential done to understand the underlying mechanism of action of the effects susceptibilities of human lung, breast and skin cancer cell lines to killing of Facial Moisturizer - Camellia & Geranium Blossom on the nuclear by five sea anemone venoms. J Venom Anim Toxins incl Trop Dis 18: division index (NDI). 157-163. 20. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Imm Met Conflict of Interest 65: 55-63. The author declares that there is no conflict of interest. 21. Fenech M (2007) Cytokinesis-block micronucleus cytome assay. Nat Protoc 2: 1084-1104. 22. Wang JJ, Sanderson BJS, Wang H (2007) Cyto- and genotoxicity of Acknowledgments ultrafine TiO2 particles in cultured human lymphoblastoid cells. Mutat Res 628: 99-106. We thank the Ministry of Education, Saudi Arabia, for partial 23. Fenech M (2000) The in vitro micronucleus technique. Mutat Res 455: support of this project. Also, we are thankful to Professor Christopher 81-95. Franco, Head of Medical Biotechnology, School of Medicine, Flinders University for valuable suggestions and reviewing the manuscript.

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J Clin Toxicol, an open access journal Volume 6 • Issue 4 • 1000315 ISSN:2161-0495